Transgenic animals are being utilized as model systems for studying both normal and disease processes. In general an exogenous gene with or without a mutation is transferred to the animal host system and the phenotype resulting from the transferred gene is observed. Other genetic manipulations can be undertaken in the vector or host system to improve the gene expression leading to the observed phenotype (phenotypic expression). The gene may be transferred under the control of different inducible or constituent promoters, may be overexpressed, the endogenous homologous gene may be rendered unexpressible and the like. PCT Application WO 92/11358; U.S. Pat. No. 5,221,778! However, even with these manipulations the desired phenotype is not always expressed (for example, see herein below regarding PS transgenes). Further, as set forth in U.S. Pat. Nos. 5,602,299 and 5,221,778 various breeding programs to change the background, insert additional transactivator transgene, make the transgene homozygous or have hosts carrying two copies of the transgene but inserted at two different sites have been disclosed. However, again these methods do not always improve the phenotypic expression of the transgene such that the model system more closely resembles the desired phenotype. Therefore additional methods are needed to improve and/or modulate the phenotype of transgenic animals.
Transgenic model systems are needed to study neurodegenerative disorders, both to understand the underlying disease pathology as well as to test treatment protocols. Alzheimer's disease (AD) is a neurodegenerative disorder with a progressive dementia characterized by the presence of extracellular amyloid deposits (composed mainly of .beta.-amyloid (A.beta.)) and intraneuronal tangles, (consisting largely of the cytoskeletal protein tau), in specific brain regions. Its symptoms include gradual memory loss, declined ability to perform routine tasks such as eating, confusion, disorientation, the inability of the patient to care for him or herself, and eventually death. The American Health Assistance Foundation has reported that presently more than four million Americans are believed to have AD, and each year 100,000 Americans die because of AD and 250,000 new cases of AD are diagnosed. Moreover, one out of every ten Americans 65 years and older have AD and almost half of those 85 years and older have the disease.
Although AD in general is associated with patients in their late 60s, 70s and older, Familial Alzheimer's Disease (FAD) has been documented in patients in their thirties and forties. FAD is genetic autosomal dominant form of AD. Although the genetic causes of FAD are not thought to be the same as AD, the FAD phenotype appears to be pathologically similar to AD. It has been reported that 10% of all AD cases are FAD.
Autopsies of patients who suffered from FAD have shown the presence of neuritic plaques made up largely of beta-amyloid (A.beta.) and neurofibrillary tangles consisting largely of deposits of tau protein reviewed in Hardy and Duff, 1993!. Beta-amyloid is a 40-42 amino acid peptide produced by the proteolytic cleavage of the larger amyloid precursor protein (APP). APP is a transmembrane protein with a single transmembrane domain running from residue 700 to residue 722. The APP gene is located on chromosome 21 and contains 18 exons. APP isoforms resulting from alternative splicing form a set of polypeptides ranging from 563 to 770 residues in length. The beta amyloid fragment is encoded by the 3' half of exon 16 and the 5' half of exon 17, which also encodes APP's transmembrane domain. Most of the beta-amyloid cleaved from APP is forty (40) amino acid residues long and designated A.beta.1-40.
Another form of beta-amyloid produced in much smaller amounts relative to the production of A.beta.1-40 is a peptide 42-43 amino acid residues long. It is designated A.beta.1-42(43). This peptide is selectively deposited early in the FAD process. Experiments conducted in vitro have demonstrated this peptide forms insoluble aggregates much faster than A.beta.1-40. Hence it is believed that increased production of A.beta.1-42(43) occurs in patients genetically predisposed to FAD and initiates its pathology. Since both forms of beta-amyloid are insoluble, they deposit on neurons in the brain.
Genetic causes of AD include mutations in the APP gene on chromosome 21, the presenilin 1 (PS1) gene on chromosome 14 and the presenilin 2 (PS2) gene on chromosome 1 Goate et al, 1991; Sherrington et al, 1995; Levy-Lahad et al, 1995!. All known mutations which cause AD have been shown to alter the processing of APP such that more amyloidogenic A.beta. (A.beta.42(43)) is generated. This had led to the hypothesis that aberrant APP processing and the generation of A.beta.42(43) may underlie the early etiopathogenesis of FAD Younkin, 1995; Scheuner, 1996; Cai et al, 1993; Citron et al, 1992; Suzuki et al, 1994!.
In some patients suffering from FAD, a missense point mutation in exon 17 at a codon 717 (transcript 770) of the APP gene was determined responsible for the increased formation of beta-amyloid, and hence FAD PCT Application PCT/GB92/00123!. Soon thereafter, other point mutations were found in the same codon in patients suffering from FAD Hardy, 1993!.
In an effort to develop animal models to study pharmaceutical agents designed to treat FAD, transgenic mice have been developed containing the missense point mutations of the human APP gene in their genome Hsiao et al., 1996; Games et al., 1995; U.S. Pat. No. 5,612,486; PCT Applications WO 92/06187; WO 93/14200; WO 96/06927!. Since a mutated APP gene is expressed in their brains, these transgenic mice have the potential to serve as models for FAD. Models with overexpression of the APP gene. (with and without mutations) have also been developed PCT Application WO 94/24266; WO 96/06927; European Patent Application EPO 653 154 A2!.
Some of these APP gene transgenic mice have been shown to produce pathology which resembles that of FAD at one year of age and older Hsiao et al., 1996 and Games et al., 1995!. The PDAPP mouse Games, et al, 1995! expresses an APP minigene with the V717F mutation (Note mutations are abbreviated as the amino acid at location followed by the substituted amino acid). The Tg2576 mouse Hsiao et al, 1996! expresses the APP695 isoform containing a K670N,M671L mutation (APP770 numbering) which is often referred to as the Swedish mutation. In addition to AD-type pathology, Tg2576 shows cognitive impairment as measured by spontaneous alternation in a "Y" maze and spatial memory in a water maze suggesting that the manipulation of APP affects cognitive function in addition to pathology.
However, other APP gene transgenic mice do not produce or have weak FAD pathology. See Neve et al, 1996 for an example of weak pathology and Hsaio et al., 1995; Andra et al., 1996; Malherbe et al., 1996; Mucke et al., 1994 for examples of APP mice which do not show pathology and see Greenberg et al, 1996 for a review of additional APP gene transgenic mice which do not show pathology or show only weak pathology.
Analysis of the above suggests that these Alzheimer's models suffer from the limitation that they are unable to produce sufficient amounts of A.beta. in the brain to initiate Alzheimer's related pathology. Therefore transgenic models producing sufficient amounts of A.beta. in the brain in an accelerated manner and methods of making such transgenics are needed.
Recently, mutations in other genes, termed the Presenilin I (PSI) and Presenilin II (PS2) genes located on chromosomes 14 and 1, respectively, have also been shown to cause FAD. Cruts, et al., 1996!. Research has demonstrated that peripheral cells from individuals with these presenilin mutations produce a greater amount A.beta.1-42(43) than that produced in individuals having a non-mutant PS gene Scheuner et al., 1996!. It has been suggested that the mode of pathogenesis produced by these mutated presenilin genes involves the production of more A.beta.1-42(43) relative to the amount produced by a nonmutant (wildtype) PS1 or PS2 gene. Presently, the mechanism which causes this increased production of A.beta.1-42(43) is not known.
Transgenic mice carrying mutations in PS1 do not appear to develop AD-type pathology but do show an elevation of A.beta.42(43) see Example herein!. This form of A.beta. is highly amyloidogenic and forms the early core of amyloid deposits in AD brain Mann et al, 1996; Jarrett et al, 1993!. Both PS1 and PS2 are known to influence APP processing Scheuner et al, 1996 and see Example herein!. Sequence homology between the presenilins and a C. elegans protein involved in protein trafficking (SPE4) suggests that the presenilins may direct the compartmentalization and trafficking of APP L'Hemault and Arduengo, 1992! and that mutant presenilins may direct APP along a pathway that results in elevated levels of A.beta.42(43). In this way the biosynthetic pathways can be considered to be interactive.
The above transgenic animals provide the current models of Alzheimer's Disease. However, as discussed herein above many of these models are incomplete, in that the full pathology seen in humans is not seen, or as seen in the PS models, no pathology just elevated levels of A.beta.1-42(43) are seen. Further, the models generally require that the mice age and the pathology is not seen until nine months and generally later Hsaio et al., 1996!. This means that the animals must be maintained for extended periods of time. The cost of maintenance makes it difficult for many investigators to use these models.
It would be useful therefore to have transgenic models which show the full range of pathology of AD at an earlier age or a selected aspect of the pathology or for that matter any other human genetically based condition. The method should allow the modulation of the phenotype resulting from the expression of the transgenes. It would further be useful to have a model for AD in which the pathology onset is earlier (accelerated). Specifically, a model in which amyloid accumulation is enhanced and accelerated.